Presynaptic activity regulates Na(+) channel distribution at the axon initial segment.

Deprivation of afferent inputs in neural circuits leads to diverse plastic changes in both pre- and postsynaptic elements that restore neural activity. The axon initial segment (AIS) is the site at which neural signals arise, and should be the most efficient site to regulate neural activity. However, none of the plasticity currently known involves the AIS. We report here that deprivation of auditory input in an avian brainstem auditory neuron leads to an increase in AIS length, thus augmenting the excitability of the neuron. The length of the AIS, defined by the distribution of voltage-gated Na(+) channels and the AIS anchoring protein, increased by 1.7 times in seven days after auditory input deprivation. This was accompanied by an increase in the whole-cell Na(+) current, membrane excitability and spontaneous firing. Our work demonstrates homeostatic regulation of the AIS, which may contribute to the maintenance of the auditory pathway after hearing loss. Furthermore, plasticity at the spike initiation site suggests a powerful pathway for refining neuronal computation in the face of strong sensory deprivation.

Activity-dependent and activity-independent development of the axon initial segment.

The axon initial segment (AIS) is the site of spike initiation in neurons. Previous studies revealed that spatial distribution of the AIS varies greatly among neurons to meet their specific needs. However, when and how this differentiation arises is unknown. Neurons in the avian nucleus laminaris (NL) are binaural coincidence detectors for sound localization and show differentiation in the distribution of the AIS, with shorter length and a more distal position from the soma with an increase in tuning frequency. We studied these characteristics of the AIS in NL neurons of the chicken during development and found that the AIS differentiates in its distribution after initial formation, and this is driven by activity-dependent and activity-independent mechanisms that differentially regulate distal and proximal boundaries of the AIS. Before hearing onset, the ankyrinG-positive AIS existed at a wide stretch of proximal axon regardless of tuning frequency, but Na+ channels were only partially distributed within the AIS. Shortly after hearing onset, Na+ channels accumulated along the entire AIS, which started shortening and relocating distally to a larger extent in neurons with higher tuning frequencies. Ablation of inner ears abolished the shortening of the AIS without affecting the position of its proximal boundary, indicating that both distal and proximal AIS boundaries are disassembled during development, and the former is dependent on afferent activity. Thus, interaction of these activity-dependent and activity-independent mechanisms determines the cell-specific distribution of the AIS in NL neurons and plays a critical role in establishing the function of sound localization circuit.

Simultaneous recording of fluorescence and electrical signals by photometric patch electrode in deep brain regions in vivo.

Despite its widespread use, high-resolution imaging with multiphoton microscopy to record neuronal signals in vivo is limited to the surface of brain tissue because of limited light penetration. Moreover, most imaging studies do not simultaneously record electrical neural activity, which is, however, crucial to understanding brain function. Accordingly, we developed a photometric patch electrode (PME) to overcome the depth limitation of optical measurements and also enable the simultaneous recording of neural electrical responses in deep brain regions. The PME recoding system uses a patch electrode to excite a fluorescent dye and to measure the fluorescence signal as a light guide, to record electrical signal, and to apply chemicals to the recorded cells locally. The optical signal was analyzed by either a spectrometer of high light sensitivity or a photomultiplier tube depending on the kinetics of the responses. We used the PME in Oregon Green BAPTA-1 AM-loaded avian auditory nuclei in vivo to monitor calcium signals and electrical responses. We demonstrated distinct response patterns in three different nuclei of the ascending auditory pathway. On acoustic stimulation, a robust calcium fluorescence response occurred in auditory cortex (field L) neurons that outlasted the electrical response. In the auditory midbrain (inferior colliculus), both responses were transient. In the brain-stem cochlear nucleus magnocellularis, calcium response seemed to be effectively suppressed by the activity of metabotropic glutamate receptors. In conclusion, the PME provides a powerful tool to study brain function in vivo at a tissue depth inaccessible to conventional imaging devices.

The cooperation of sustained and phasic inhibitions increases the contrast of ITD-tuning in low-frequency neurons of the chick nucleus laminaris.

Neurons in the nucleus laminaris (NL) of birds detect the coincidence of binaural excitatory inputs from the nucleus magnocellularis (NM) on both sides and process the interaural time differences (ITDs) for sound localization. Sustained inhibition from the superior olivary nucleus is known to control the gain of coincidence detection, which allows the sensitivity of NL neurons to ITD tolerate strong-intensity sound. Here, we found a phasic inhibition in chicken brain slices that follows the ipsilateral NM inputs after a short time delay, sharpens coincidence detection, and may enhance ITD sensitivity in low-frequency NL neurons. GABA-positive small neurons are distributed in and near the NL. These neurons generate IPSCs in NL neurons when photoactivated by a caged glutamate compound, suggesting that these GABAergic neurons are interneurons that mediate phasic inhibition. These IPSCs have fast decay kinetics that is attributable to the α1-subunit of the GABAA receptor, the expression of which dominates in the low-frequency region of the NL. Model simulations demonstrate that phasic IPSCs narrow the time window of coincidence detection and increase the contrast of ITD-tuning during low-level, low-frequency excitatory input. Furthermore, cooperation of the phasic and sustained inhibitions effectively increases the contrast of ITD-tuning over a wide range of excitatory input levels. We propose that the complementary interaction between phasic and sustained inhibitions is the neural mechanism that regulates ITD sensitivity for low-frequency sound in the NL.

Activation of metabotropic glutamate receptors improves the accuracy of coincidence detection by presynaptic mechanisms in the nucleus laminaris of the chick.

Interaural time difference (ITD) is a major cue for localizing a sound source and is processed in the nucleus laminaris (NL) in birds. Coincidence detection (CD) is a crucial step for processing ITD and critically depends on the size and time course of excitatory postsynaptic potentials (EPSPs). Here, we investigated a role of metabotropic glutamate receptors (mGluRs) in the regulation of EPSP amplitude and CD in the NL of chicks. A non-specific agonist of mGluRs ((±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid; t-ACPD) reduced the amplitude and extent of depression of excitatory postsynaptic currents (EPSCs) during a stimulus train, while the paired pulse ratio and coefficient of variation of EPSC amplitude were increased. In contrast, the amplitudes of spontaneous EPSCs were not affected, but the frequency was reduced. Thus, the effects of t-ACPD were presynaptic and reduced the release of neurotransmitter from terminals in the NL. Expression of group II mGluRs was graded along the tonotopic axis and was stronger towards the low frequency region in the NL. Both group II (DCG-IV) and group III (l-AP4) specific agonists reduced EPSC amplitude by presynaptic mechanisms, and the reduction was larger in the low frequency region; however, we could not find any effects of group I-specific agonists on EPSCs. The reduced EPSP amplitude in DCG-IV improved CD. A specific antagonist of group II mGluRs (LY341495) increased the amplitude of both EPSCs and EPSPs and enhanced the depression during a stimulus train, indicating constitutive activation of mGluRs in the NL. These observations indicate that mGluRs may work as autoreceptors and regulate EPSP size to improve CD in the NL.

Hyperpolarisation-activated cyclic nucleotide-gated channels regulate the spontaneous firing rate of olfactory receptor neurons and affect glomerular formation in mice.

Olfactory receptor neurons (ORNs), which undergo lifelong neurogenesis, have been studied extensively to understand how neurons form precise topographical networks. Neural projections from ORNs are principally guided by the genetic code, which directs projections from ORNs that express a specific odorant receptor to the corresponding glomerulus in the olfactory bulb. In addition, ORNs utilise spontaneous firing activity to establish and maintain the neural map. However, neither the process of generating this spontaneous activity nor its role as a guidance cue in the olfactory bulb is clearly understood. Utilising extracellular unit-recordings in mouse olfactory epithelium slices, we demonstrated that the hyperpolarisation-activated cyclic nucleotide-gated (HCN) channels in the somas of ORNs depolarise their membranes and boost their spontaneous firing rates by sensing basal cAMP levels; the odorant-sensitive cyclic nucleotide-gated (CNG) channels in cilia do not. The basal cAMP levels were maintained via the standing activation of ß-adrenergic receptors. Using a Tet-off system to over-express HCN4 channels resulted in the enhancement of spontaneous ORN activity and dramatically reduced both the size and number of glomeruli in the olfactory bulb. This phenotype was rescued by the administration of doxycycline. These findings suggest that cAMP plays different roles in cilia and soma and that basal cAMP levels in the soma are directly converted via HCN channels into a spontaneous firing frequency that acts as an intrinsic guidance cue for the formation of olfactory networks.

Convergence of bilateral auditory midbrain inputs on neurons in the auditory thalamus of chicken.

In the avian ascending auditory pathway, the nucleus mesencephalicus lateralis pars dorsalis (MLd; the auditory midbrain center) receives inputs from virtually all lower brainstem auditory nuclei and sends outputs bilaterally to the nucleus ovoidalis (Ov; the auditory thalamic nucleus). Axons from part of the MLd terminate in a particular domain of Ov, thereby suggesting a formation of segregated pathways point-to-point from lower brainstem nuclei via MLd to the thalamus. However, it has not yet been demonstrated whether any spatial clustering of thalamic neurons that receive inputs from specific domains of MLd exists. Ov neurons receive input from bilateral MLds; however, the degree of laterality has not been reported yet. In this study, we injected a recombinant avian adeno-associated virus, a transsynaptic anterograde vector into the MLd of the chick, and analyzed the distribution of labeled postsynaptic neurons on both sides of the Ov. We found that fluorescent protein-labeled neurons on both sides of the Ov were clustered in domains corresponding to subregions of the MLd. The laterality of projections was calculated as the ratio of neurons labeled by comparing ipsilateral to contralateral projections from the MLd, and it was 1.86 on average, thereby indicating a slight ipsilateral projection dominance. Bilateral inputs from different subdomains of the MLd converged on several single Ov neurons, thereby implying a possibility of a de novo binaural processing of the auditory information in the Ov.

High resolution recording of local field currents simultaneously with sound-evoked calcium signals by a photometric patch electrode in the auditory cortex field L of the chick.

BACKGROUND: Functioning of the brain is based on both electrical and metabolic activity of neural ensembles. Accordingly, it would be useful to measure intracellular metabolic signaling simultaneously with electrical activity in the brain in vivo. NEW METHOD: We innovated a PhotoMetric-patch-Electrode (PME) recording system that has a high temporal resolution incorporating a photomultiplier tube as a light detector. The PME is fabricated from a quartz glass capillary to transmit light as a light guide, and it can detect electrical signals as a patch electrode simultaneously with a fluorescence signal. RESULTS: We measured the sound-evoked Local Field Current (LFC) and fluorescence Ca2+ signal from neurons labeled with Ca2+-sensitive dye Oregon Green BAPTA1 in field L, the avian auditory cortex. Sound stimulation evoked multi-unit spike bursts and Ca2+ signals, and enhanced the fluctuation of LFC. After a brief sound stimulation, the cross-correlation between LFC and Ca2+ signal was prolonged. D-AP5 (antagonist for NMDA receptors) suppressed the sound-evoked Ca2+ signal when applied locally by pressure from the tip of PME. COMPARISON WITH EXISTING METHODS: In contrast to existing multiphoton imaging or optical fiber recording methods, the PME is a patch electrode pulled simply from a quartz glass capillary and can measure fluorescence signals at the tip simultaneously with electrical signal at any depth of the brain structure. CONCLUSION: The PME is devised to record electrical and optical signals simultaneously with high temporal resolution. Moreover, it can inject chemical agents dissolved in the tip-filling medium locally by pressure, allowing manipulation of neural activity pharmacologically.

Axonal site of spike initiation enhances auditory coincidence detection.

Neurons initiate spikes in the axon initial segment or at the first node in the axon. However, it is not yet understood how the site of spike initiation affects neuronal activity and function. In nucleus laminaris of birds, neurons behave as coincidence detectors for sound source localization and encode interaural time differences (ITDs) separately at each characteristic frequency (CF). Here we show, in nucleus laminaris of the chick, that the site of spike initiation in the axon is arranged at a distance from the soma, so as to achieve the highest ITD sensitivity at each CF. Na+ channels were not found in the soma of high-CF (2.5-3.3 kHz) and middle-CF (1.0-2.5 kHz) neurons but were clustered within a short segment of the axon separated by 20-50 microm from the soma; in low-CF (0.4-1.0 kHz) neurons they were clustered in a longer stretch of the axon closer to the soma. Thus, neurons initiate spikes at a more remote site as the CF of neurons increases. Consequently, the somatic amplitudes of both orthodromic and antidromic spikes were small in high-CF and middle-CF neurons and were large in low-CF neurons. Computer simulation showed that the geometry of the initiation site was optimized to reduce the threshold of spike generation and to increase the ITD sensitivity at each CF. Especially in high-CF neurons, a distant localization of the spike initiation site improved the ITD sensitivity because of electrical isolation of the initiation site from the soma and dendrites, and because of reduction of Na+-channel inactivation by attenuating the temporal summation of synaptic potentials through the low-pass filtering along the axon.

A light-induced small G-protein gem limits the circadian clock phase-shift magnitude by inhibiting voltage-dependent calcium channels.

Calcium signaling is pivotal to the circadian clockwork in the suprachiasmatic nucleus (SCN), particularly in rhythm entrainment to environmental light-dark cycles. Here, we show that a small G-protein Gem, an endogenous inhibitor of high-voltage-activated voltage-dependent calcium channels (VDCCs), is rapidly induced by light in SCN neurons via the calcium (Ca2+)-mediated CREB/CRE transcriptional pathway. Gem attenuates light-induced calcium signaling through its interaction with VDCCs. The phase-shift magnitude of locomotor activity rhythms by light, at night, increases in Gem-deficient (Gem-/-) mice. Similarly, in SCN slices from Gem-/- mice, depolarizing stimuli induce larger phase shifts of clock gene transcription rhythms that are normalized by the application of an L-type VDCC blocker, nifedipine. Voltage-clamp recordings from SCN neurons reveal that Ca2+ currents through L-type channels increase in Gem-/- mice. Our findings suggest that transcriptionally activated Gem feeds back to suppress excessive light-evoked L-type VDCC activation, adjusting the light-induced phase-shift magnitude to an appropriate level in mammals.

GABAergic inhibition sharpens the frequency tuning and enhances phase locking in chicken nucleus magnocellularis neurons.

GABAergic modulation of activity in avian cochlear nucleus neurons has been studied extensively in vitro. However, how this modulation actually influences processing in vivo is not known. We investigated responses of chicken nucleus magnocellularis (NM) neurons to sound while pharmacologically manipulating the inhibitory input from the superior olivary nucleus (SON). SON receives excitatory inputs from nucleus angularis (NA) and nucleus laminaris (NL), and provides GABAergic inputs to NM, NA, NL, and putatively to the contralateral SON. Results from single-unit extracellular recordings from 2 to 4 weeks posthatch chickens show that firing rates of auditory nerve fibers increased monotonically with sound intensity, while that of NM neurons saturated or even decreased at moderate or loud sound levels. Blocking GABAergic input with local application of TTX into the SON induced an increase in firing rate of ipsilateral NM, while that of the contralateral NM decreased at high sound levels. Moreover, local application of bicuculline to NM also increased the firing rate of NM neurons at high sound levels, reduced phase locking, and broadened the frequency-tuning properties of NM neurons. Following application of DNQX, clear evidence of inhibition was observed. Furthermore, the inhibition was tuned to a broader frequency range than the excitatory response areas. We conclude that GABAergic inhibition from SON has at least three physiological influences on the activity of NM neurons: it regulates the firing activity of NM units in a sound-level-dependent manner; it improves phase selectivity; and it sharpens frequency tuning of NM neuronal responses.

Inhibition in the balance: binaurally coupled inhibitory feedback in sound localization circuitry.

Interaural time differences (ITDs) are the primary cue animals, including humans, use to localize low-frequency sounds. In vertebrate auditory systems, dedicated ITD processing neural circuitry performs an exacting task, the discrimination of microsecond differences in stimulus arrival time at the two ears by coincidence-detecting neurons. These neurons modulate responses over their entire dynamic range to sounds differing in ITD by mere hundreds of microseconds. The well-understood function of this circuitry in birds has provided a fruitful system to investigate how inhibition contributes to neural computation at the synaptic, cellular, and systems level. Our recent studies in the chicken have made significant progress in bringing together many of these findings to provide a cohesive picture of inhibitory function.

Avian adeno-associated virus as an anterograde transsynaptic vector.

BACKGROUND: Retrograde and anterograde transsynaptic viral vectors are useful tools for studying the input and output organization of neuronal circuitry, respectively. While retrograde transsynaptic viral vectors are widely used, viral vectors that show anterograde transsynaptic transduction are not common. NEW METHOD: We chose recombinant avian adeno-associated virus (A3V) carrying the mCherry gene and injected it into the eyeball, cochlear duct, and midbrain auditory center of chickens. We observed different survival times to examine the virus transcellular transport and the resulting mCherry expression. To confirm the transcellular transduction mode, we co-injected A3V and cholera toxin B subunit. RESULTS: Injecting A3V into the eyeball and cochlea labeled neurons in the visual and auditory pathways, respectively. Second-, and third-order labeling occurred approximately two and seven days, respectively, after injection into the midbrain. The distribution of labeled neurons strongly suggests that A3V transport is preferentially anterograde and transduces postsynaptic neurons. COMPARISON WITH EXISTING METHOD(S): A3V displays no extrasynaptic leakage and moderate speed of synapse passage, which is better than other viruses previously reported. Compared with AAV1&9, which have been shown to pass one synapse anterogradely, A3V passes several synapses in the anterograde direction. CONCLUSIONS: A3V would be a good tool to study the topographic organization of projection axons and their target neurons.

Olfactory marker protein directly buffers cAMP to avoid depolarization-induced silencing of olfactory receptor neurons.

Olfactory receptor neurons (ORNs) use odour-induced intracellular cAMP surge to gate cyclic nucleotide-gated nonselective cation (CNG) channels in cilia. Prolonged exposure to cAMP causes calmodulin-dependent feedback-adaptation of CNG channels and attenuates neural responses. On the other hand, the odour-source searching behaviour requires ORNs to be sensitive to odours when approaching targets. How ORNs accommodate these conflicting aspects of cAMP responses remains unknown. Here, we discover that olfactory marker protein (OMP) is a major cAMP buffer that maintains the sensitivity of ORNs. Upon the application of sensory stimuli, OMP directly captured and swiftly reduced freely available cAMP, which transiently uncoupled downstream CNG channel activity and prevented persistent depolarization. Under repetitive stimulation, OMP-/- ORNs were immediately silenced after burst firing due to sustained depolarization and inactivated firing machinery. Consequently, OMP-/- mice showed serious impairment in odour-source searching tasks. Therefore, cAMP buffering by OMP maintains the resilient firing of ORNs.

Sound-intensity-dependent compensation for the small interaural time difference cue for sound source localization.

Interaural time difference (ITD) is a major cue for sound source localization. However, animals with small heads experience small ITDs, making ITD detection difficult, particularly for low-frequency sound. Here, we describe a sound-intensity-dependent mechanism for compensating for the small ITD cues in the coincidence detector neurons in the nucleus laminaris (NL) of the chicken aged from 3 to 29 d after hatching. The hypothesized compensation mechanisms were confirmed by simulation. In vivo single-unit recordings revealed an improved contrast of ITD tuning in low-best-frequency (<1 kHz) NL neurons by suppressing the firing activity at the worst ITD, whereas the firing rate was increased with increasing sound intensity at the best ITD. In contrast, level-dependent suppression was so weak in the middle- and high-best-frequency (> or =1 kHz) NL neurons that loud sounds led to increases in firing rate at both the best and the worst ITDs. The suppression of firing activity at the worst ITD in the low-best-frequency neurons required the activation of the superior olivary nucleus (SON) and was eliminated by electrolytic lesions of the SON. The frequency-dependent suppression reflected the dense projection from the SON to the low-frequency region of NL. Thus, the small ITD cues available in low-frequency sounds were partly compensated for by a sound-intensity-dependent inhibition from the SON.

Roles of axonal sodium channels in precise auditory time coding at nucleus magnocellularis of the chick.

How the axonal distribution of Na(+) channels affects the precision of spike timing is not well understood. We addressed this question in auditory relay neurons of the avian nucleus magnocellularis. These neurons encode and convey information about the fine structure of sounds to which they are tuned by generating precisely timed action potentials in response to synaptic inputs. Patterns of synaptic inputs differ as a function of tuning. A small number of large inputs innervate high- and middle-frequency neurons, while a large number of small inputs innervate low-frequency neurons. We found that the distribution and density of Na(+) channels in the axon initial segments varied with the synaptic inputs, and were distinct in the low-frequency neurons. Low-frequency neurons had a higher density of Na(+) channels within a longer axonal stretch, and showed a larger spike amplitude and whole-cell Na(+) current than high/middle-frequency neurons. Computer simulations revealed that for low-frequency neurons, a large number of Na(+) channels were crucial for preserving spike timing because it overcame Na(+) current inactivation and K(+) current activation during compound EPSPs evoked by converging small inputs. In contrast, fewer channels were sufficient to generate a spike with high precision in response to an EPSP induced by a single massive input in the high/middle-frequency neurons. Thus the axonal Na(+) channel distribution is effectively coupled with synaptic inputs, allowing these neurons to convey auditory information in the timing of firing.

The modulation by intensity of the processing of interaural timing cues for localizing sounds.

Features of sounds such as time and intensity are important binaural cues for localizing their sources. Interaural time differences (ITDs) and interaural level differences are extracted and processed in parallel by separate pathways in the brainstem auditory nuclei. ITD cues are small, particularly in small-headed animals, and processing of these cues is optimized by both morphological and physiological specializations. Moreover, recent observations in mammals and in some birds indicate that interaural time and level cues are not processed independently but cooperatively to improve the detection of interaural differences. This review will specifically summarize what is known about how inhibitory circuits improve the measurements of ITD in a sound-level-dependent manner.

Presynaptic GABA(B) receptors modulate synaptic facilitation and depression at distinct synapses in fusiform cells of mouse dorsal cochlear nucleus.

The mammalian dorsal cochlear nucleus (DCN) is considered to contribute to the localization of the sound sources. Fusiform cells (FCs), principal projection neurons in the DCN, integrate two excitatory inputs from auditory nerve fibers (ANFs) and parallel fibers (PFs). Although an immunohistochemical study suggested presence of GABA(B) receptors at excitatory presynaptic terminals in the DCN, it has not been elucidated how GABA(B) receptors modulate the synaptic transmission to FCs. Here, we examined effects of baclofen on the transmission in vitro. Baclofen reduced both PF-EPSC and ANF-EPSC by reducing transmitter releases, and it enhanced the facilitation in PF-FC synapses and prevented the depression in ANF-FC synapses. The enhancement and prevention were prominent during high-frequency (50Hz) synaptic input, suggesting the activation of presynaptic GABA(B) receptors may optimize both PF-FC and ANF-FC synapses for high-frequency transmission. Postsynaptic GABA(B) receptors activated GIRK current and would further modulate the activity of FCs.

Tonotopic specialization of auditory coincidence detection in nucleus laminaris of the chick.

The interaural time difference (ITD) is a cue for localizing a sound source along the horizontal plane and is first determined in the nucleus laminaris (NL) in birds. Neurons in NL are tonotopically organized, such that ITDs are processed separately at each characteristic frequency (CF). Here, we investigated the excitability and coincidence detection of neurons along the tonotopic axis in NL, using a chick brainstem slice preparation. Systematic changes with CF were observed in morphological and electrophysiological properties of NL neurons. These properties included the length of dendrites, the input capacitance, the conductance of hyperpolarization-activated current, and the EPSC time course. In contrast to these gradients, the conductance of low-threshold K+ current and the expression of Kv1.2 channel protein were maximal in the central (middle-CF) region of NL. As a result, the middle-CF neuron had the smallest input resistance and membrane time constant, and consequently the fastest EPSP, and exhibited the most accurate coincidence detection. The specialization of middle-CF neurons as coincidence detectors may account for the high resolution of sound-source localization in the middle-frequency range observed in avians.

Hyperpolarization-activated cyclic nucleotide-gated cation channels regulate auditory coincidence detection in nucleus laminaris of the chick.

Coincidence detection of bilateral acoustic signals in nucleus laminaris (NL) is the first step in azimuthal sound source localization in birds. Here, we demonstrate graded expression of hyperpolarization-activated cyclic nucleotide-gated (HCN) cation channels along the tonotopic axis of NL and its role in the regulation of coincidence detection. Expression of HCN1 and HCN2, but not HCN3 or HCN4, was detected in NL. Based on measurement of both subtype mRNA and protein, HCN1 varied along the tonotopic axis and was minimal in high-characteristic frequency (CF) neurons. In contrast, HCN2 was evenly distributed. The resting conductance was larger and the steady-state activation curve of Ih was more positive in neurons of middle to low CF than those of high CF, consistent with the predominance of HCN1 channels in these neurons. Application of 8-Br-cAMP or noradrenaline generated a depolarizing shift of the Ih voltage activation curve. This shift was larger in neurons of high CF than in those of middle CF. The shift in the activation voltage of Ih depolarized the resting membrane, accelerated the EPSP time course, and significantly improved the coincidence detection in neurons of high CF, suggesting that Ih may improve the localization of sound sources.